Manual magnetic-bead total RNA purification from low-yield samples
Cat. No. IVD3020B | DNase I on-bead treatment | sample-specific cumulative timing estimates
Use no more than 10 mg animal tissue. Transfer the tissue to a 1.5 ml centrifuge tube, add 500 µl Buffer RLC and disperse the sample thoroughly. Centrifuge at 14,000 × g for 3 min at room temperature and use the clarified lysate or supernatant for binding.
The first step includes sample dispersion and the 3 min clarification spin. Incomplete dispersion at this stage is a common cause of low RNA recovery.
Add 20 µl MagPure Particles N and 500 µl Buffer MCB to a clean 1.5 ml centrifuge tube. Mix the particle suspension before pipetting so that the bead input remains uniform.
Buffer MCB must be supplemented with isopropanol before use.
Transfer 500 µl prepared lysate or clarified supernatant into the binding tube. Mix by inverting 15–20 times, incubate at room temperature for 10 min, invert several times during incubation, then place on a magnetic rack for about 2 min and remove the supernatant.
This step establishes the alcohol / salt condition for RNA adsorption onto silica magnetic particles.
Add 500 µl Buffer GW1 and vortex for 10 s to fully resuspend the particles. Place the tube on the magnetic rack for about 1 min and remove the supernatant.
Remove wash liquid carefully without disturbing the bead pellet.
Add 500 µl freshly prepared DNase Mixture to the bead pellet. Prepare the mixture as 490 µl DNase Buffer C plus 10 µl DNase I per sample. Mix by gentle oscillation and incubate at room temperature for 15 min, then place on the magnetic rack for about 1 min and remove the supernatant.
This DNase step is part of the standard IVD3020B route and is used to reduce genomic DNA carryover.
Add 500 µl Buffer GW1, vortex for 10 s to resuspend the particles, place on the magnetic rack for about 1 min and remove the supernatant.
This wash removes DNase reaction components before the ethanol-based MW2 washes.
Add 500 µl Buffer MW2, vortex for 10 s to resuspend the particles, place on the magnetic rack for about 1 min and remove the supernatant.
Buffer MW2 must be supplemented with ethanol before use.
Repeat the MW2 wash once using 500 µl Buffer MW2. Magnetically separate the particles for about 1 min and remove the supernatant.
A second MW2 wash helps remove residual salt and alcohol-soluble impurities.
Briefly centrifuge the tube to collect residual liquid, return it to the magnetic rack if needed and remove all remaining wash liquid carefully. Air dry the particles for about 10–15 min.
The range reflects the protocol drying window plus careful residual-liquid removal. Avoid over-drying the beads.
Add 30–100 µl RNase-free Water to the sample, vortex to resuspend the particles completely and incubate at room temperature for 3 min.
Use a smaller elution volume when RNA concentration is more important than total recovered volume.
Place the tube on the magnetic rack for about 3 min until the eluate is clear. Transfer the supernatant containing purified RNA to a new RNase-free 1.5 ml tube and store at -20°C or -80°C.
Do not carry magnetic particles into the final RNA eluate.
Use no more than 30 mg plant tissue. Grind the sample to a fine powder in liquid nitrogen, transfer the powder to a 1.5 ml tube, add 500 µl Buffer RLC immediately, vortex for 10 s and centrifuge at 13,000 × g for 3 min at room temperature. Use the clarified supernatant for binding.
For polysaccharide- or polyphenol-rich plant material, replace RLC with Buffer RLF. For starch-rich samples prone to gelatinization, use the PAL / phenol-chloroform route according to the product insert.
Add 20 µl MagPure Particles N and 500 µl Buffer MCB to a clean 1.5 ml centrifuge tube. Mix the particle suspension before pipetting so that the bead input remains uniform.
Buffer MCB must be supplemented with isopropanol before use.
Transfer 500 µl prepared lysate or clarified supernatant into the binding tube. Mix by inverting 15–20 times, incubate at room temperature for 10 min, invert several times during incubation, then place on a magnetic rack for about 2 min and remove the supernatant.
This step establishes the alcohol / salt condition for RNA adsorption onto silica magnetic particles.
Add 500 µl Buffer GW1 and vortex for 10 s to fully resuspend the particles. Place the tube on the magnetic rack for about 1 min and remove the supernatant.
Remove wash liquid carefully without disturbing the bead pellet.
Add 500 µl freshly prepared DNase Mixture to the bead pellet. Prepare the mixture as 490 µl DNase Buffer C plus 10 µl DNase I per sample. Mix by gentle oscillation and incubate at room temperature for 15 min, then place on the magnetic rack for about 1 min and remove the supernatant.
This DNase step is part of the standard IVD3020B route and is used to reduce genomic DNA carryover.
Add 500 µl Buffer GW1, vortex for 10 s to resuspend the particles, place on the magnetic rack for about 1 min and remove the supernatant.
This wash removes DNase reaction components before the ethanol-based MW2 washes.
Add 500 µl Buffer MW2, vortex for 10 s to resuspend the particles, place on the magnetic rack for about 1 min and remove the supernatant.
Buffer MW2 must be supplemented with ethanol before use.
Repeat the MW2 wash once using 500 µl Buffer MW2. Magnetically separate the particles for about 1 min and remove the supernatant.
A second MW2 wash helps remove residual salt and alcohol-soluble impurities.
Briefly centrifuge the tube to collect residual liquid, return it to the magnetic rack if needed and remove all remaining wash liquid carefully. Air dry the particles for about 10–15 min.
The range reflects the protocol drying window plus careful residual-liquid removal. Avoid over-drying the beads.
Add 30–100 µl RNase-free Water to the sample, vortex to resuspend the particles completely and incubate at room temperature for 3 min.
Use a smaller elution volume when RNA concentration is more important than total recovered volume.
Place the tube on the magnetic rack for about 3 min until the eluate is clear. Transfer the supernatant containing purified RNA to a new RNase-free 1.5 ml tube and store at -20°C or -80°C.
Do not carry magnetic particles into the final RNA eluate.
Use no more than 3 × 10⁶ suspension cells. Transfer the cell culture to a centrifuge tube, centrifuge at 500 × g for 10 min to collect cells, remove the supernatant completely, loosen the pellet, add 500 µl Buffer RLC and pipette several times to mix.
If the lysate is viscous, pass it 3–5 times through a 1 ml syringe before magnetic binding to reduce viscosity and improve handling.
Add 20 µl MagPure Particles N and 500 µl Buffer MCB to a clean 1.5 ml centrifuge tube. Mix the particle suspension before pipetting so that the bead input remains uniform.
Buffer MCB must be supplemented with isopropanol before use.
Transfer 500 µl prepared lysate or clarified supernatant into the binding tube. Mix by inverting 15–20 times, incubate at room temperature for 10 min, invert several times during incubation, then place on a magnetic rack for about 2 min and remove the supernatant.
This step establishes the alcohol / salt condition for RNA adsorption onto silica magnetic particles.
Add 500 µl Buffer GW1 and vortex for 10 s to fully resuspend the particles. Place the tube on the magnetic rack for about 1 min and remove the supernatant.
Remove wash liquid carefully without disturbing the bead pellet.
Add 500 µl freshly prepared DNase Mixture to the bead pellet. Prepare the mixture as 490 µl DNase Buffer C plus 10 µl DNase I per sample. Mix by gentle oscillation and incubate at room temperature for 15 min, then place on the magnetic rack for about 1 min and remove the supernatant.
This DNase step is part of the standard IVD3020B route and is used to reduce genomic DNA carryover.
Add 500 µl Buffer GW1, vortex for 10 s to resuspend the particles, place on the magnetic rack for about 1 min and remove the supernatant.
This wash removes DNase reaction components before the ethanol-based MW2 washes.
Add 500 µl Buffer MW2, vortex for 10 s to resuspend the particles, place on the magnetic rack for about 1 min and remove the supernatant.
Buffer MW2 must be supplemented with ethanol before use.
Repeat the MW2 wash once using 500 µl Buffer MW2. Magnetically separate the particles for about 1 min and remove the supernatant.
A second MW2 wash helps remove residual salt and alcohol-soluble impurities.
Briefly centrifuge the tube to collect residual liquid, return it to the magnetic rack if needed and remove all remaining wash liquid carefully. Air dry the particles for about 10–15 min.
The range reflects the protocol drying window plus careful residual-liquid removal. Avoid over-drying the beads.
Add 30–100 µl RNase-free Water to the sample, vortex to resuspend the particles completely and incubate at room temperature for 3 min.
Use a smaller elution volume when RNA concentration is more important than total recovered volume.
Place the tube on the magnetic rack for about 3 min until the eluate is clear. Transfer the supernatant containing purified RNA to a new RNase-free 1.5 ml tube and store at -20°C or -80°C.
Do not carry magnetic particles into the final RNA eluate.
Use no more than 3 × 10⁶ adherent cells. Remove the culture medium, add 500 µl Buffer RLC directly to the culture dish, pipette several times to detach and lyse the cells, then transfer the lysate to a 1.5 ml centrifuge tube.
If the lysate is viscous, pipette repeatedly or pass it 3–5 times through a 1 ml syringe before magnetic binding.
Add 20 µl MagPure Particles N and 500 µl Buffer MCB to a clean 1.5 ml centrifuge tube. Mix the particle suspension before pipetting so that the bead input remains uniform.
Buffer MCB must be supplemented with isopropanol before use.
Transfer 500 µl prepared lysate or clarified supernatant into the binding tube. Mix by inverting 15–20 times, incubate at room temperature for 10 min, invert several times during incubation, then place on a magnetic rack for about 2 min and remove the supernatant.
This step establishes the alcohol / salt condition for RNA adsorption onto silica magnetic particles.
Add 500 µl Buffer GW1 and vortex for 10 s to fully resuspend the particles. Place the tube on the magnetic rack for about 1 min and remove the supernatant.
Remove wash liquid carefully without disturbing the bead pellet.
Add 500 µl freshly prepared DNase Mixture to the bead pellet. Prepare the mixture as 490 µl DNase Buffer C plus 10 µl DNase I per sample. Mix by gentle oscillation and incubate at room temperature for 15 min, then place on the magnetic rack for about 1 min and remove the supernatant.
This DNase step is part of the standard IVD3020B route and is used to reduce genomic DNA carryover.
Add 500 µl Buffer GW1, vortex for 10 s to resuspend the particles, place on the magnetic rack for about 1 min and remove the supernatant.
This wash removes DNase reaction components before the ethanol-based MW2 washes.
Add 500 µl Buffer MW2, vortex for 10 s to resuspend the particles, place on the magnetic rack for about 1 min and remove the supernatant.
Buffer MW2 must be supplemented with ethanol before use.
Repeat the MW2 wash once using 500 µl Buffer MW2. Magnetically separate the particles for about 1 min and remove the supernatant.
A second MW2 wash helps remove residual salt and alcohol-soluble impurities.
Briefly centrifuge the tube to collect residual liquid, return it to the magnetic rack if needed and remove all remaining wash liquid carefully. Air dry the particles for about 10–15 min.
The range reflects the protocol drying window plus careful residual-liquid removal. Avoid over-drying the beads.
Add 30–100 µl RNase-free Water to the sample, vortex to resuspend the particles completely and incubate at room temperature for 3 min.
Use a smaller elution volume when RNA concentration is more important than total recovered volume.
Place the tube on the magnetic rack for about 3 min until the eluate is clear. Transfer the supernatant containing purified RNA to a new RNase-free 1.5 ml tube and store at -20°C or -80°C.
Do not carry magnetic particles into the final RNA eluate.
For 0.5–1.0 ml fresh blood or a bone marrow / fresh blood mixture, first prepare leukocyte cells using RBC Buffer as described in the product insert. Discard the supernatant, leave about 30 µl liquid, resuspend the leukocyte pellet completely, add 500 µl Buffer RLC and pipette several times to lyse.
RBC Buffer is supplied separately. If the lysate is too viscous, pass it 3–5 times through a 1 ml syringe to reduce viscosity before magnetic binding.
Add 20 µl MagPure Particles N and 500 µl Buffer MCB to a clean 1.5 ml centrifuge tube. Mix the particle suspension before pipetting so that the bead input remains uniform.
Buffer MCB must be supplemented with isopropanol before use.
Transfer 500 µl prepared lysate or clarified supernatant into the binding tube. Mix by inverting 15–20 times, incubate at room temperature for 10 min, invert several times during incubation, then place on a magnetic rack for about 2 min and remove the supernatant.
This step establishes the alcohol / salt condition for RNA adsorption onto silica magnetic particles.
Add 500 µl Buffer GW1 and vortex for 10 s to fully resuspend the particles. Place the tube on the magnetic rack for about 1 min and remove the supernatant.
Remove wash liquid carefully without disturbing the bead pellet.
Add 500 µl freshly prepared DNase Mixture to the bead pellet. Prepare the mixture as 490 µl DNase Buffer C plus 10 µl DNase I per sample. Mix by gentle oscillation and incubate at room temperature for 15 min, then place on the magnetic rack for about 1 min and remove the supernatant.
This DNase step is part of the standard IVD3020B route and is used to reduce genomic DNA carryover.
Add 500 µl Buffer GW1, vortex for 10 s to resuspend the particles, place on the magnetic rack for about 1 min and remove the supernatant.
This wash removes DNase reaction components before the ethanol-based MW2 washes.
Add 500 µl Buffer MW2, vortex for 10 s to resuspend the particles, place on the magnetic rack for about 1 min and remove the supernatant.
Buffer MW2 must be supplemented with ethanol before use.
Repeat the MW2 wash once using 500 µl Buffer MW2. Magnetically separate the particles for about 1 min and remove the supernatant.
A second MW2 wash helps remove residual salt and alcohol-soluble impurities.
Briefly centrifuge the tube to collect residual liquid, return it to the magnetic rack if needed and remove all remaining wash liquid carefully. Air dry the particles for about 10–15 min.
The range reflects the protocol drying window plus careful residual-liquid removal. Avoid over-drying the beads.
Add 30–100 µl RNase-free Water to the sample, vortex to resuspend the particles completely and incubate at room temperature for 3 min.
Use a smaller elution volume when RNA concentration is more important than total recovered volume.
Place the tube on the magnetic rack for about 3 min until the eluate is clear. Transfer the supernatant containing purified RNA to a new RNase-free 1.5 ml tube and store at -20°C or -80°C.
Do not carry magnetic particles into the final RNA eluate.
This workflow separates sample-specific Buffer RLC lysis / clarification from the shared magnetic silica-particle purification route. It is intended as a practical companion to the product manual rather than a replacement for the official protocol. Animal tissue and plant tissue enter the workflow after lysis and clarification centrifugation, cultured cells enter after direct lysis, and blood / bone marrow samples enter after leukocyte enrichment by red blood cell lysis. The downstream steps follow an MCB-adjusted RNA-binding, MagPure Particles N capture, magnetic separation, GW1 wash, on-bead DNase I digestion, MW2 wash, controlled drying and RNase-free water elution workflow. Because sample-entry burden differs by matrix, each sample panel shows its own running cumulative estimate, while the final total is reported as a sample-specific time range.
| Sample route | Displayed preparation estimate | Typical manual workflow time |
|---|---|---|
| Animal Tissue | 8 min | 65–85 min |
| Plant Tissue | 12 min | 70–90 min |
| Suspension Cells | 15 min | 72–92 min |
| Adherent Cells | 7 min | 64–83 min |
| Blood / Bone Marrow | 20 min | 75–100 min |
Protocol times stated in the product manual are retained where applicable. Steps without explicit timing are estimated for an experienced operator, including pipetting, tube transfer, centrifuge handling, magnetic-rack placement, bead resuspension, supernatant removal, short spin, residual-liquid removal, drying control, elution and final tube transfer. For short protocol ranges, the timeline uses the midpoint or a near-midpoint value. For long or optional protocol ranges, the displayed standard timeline uses the shortest reasonable path, while the note and total-time range indicate where extended handling may apply. For this IVD3020B workflow, the main timing variables are sample-entry difficulty, lysate viscosity, bead resuspension, residual-liquid removal and the 10–15 min drying window.
IVD3020B uses Buffer RLC lysis followed by MCB-adjusted magnetic silica-particle binding, with DNase I digestion performed directly on the bead-bound RNA. The workflow therefore depends on efficient lysate clarification, uniform MagPure Particles N resuspension and complete magnetic separation before each liquid-removal step.
The most important control points are sample input, fraction selection and bead handling. Do not overload the sample, reduce viscosity before binding, recover the correct leukocyte pellet or clarified supernatant according to sample type, and keep the particles fully resuspended during binding, DNase digestion and elution. After MW2 washing, remove residual liquid carefully and dry within the recommended window; residual ethanol can inhibit downstream reactions, while over-drying can make the beads difficult to resuspend.